Primary rat hepatocyte cultures can undergo DNA synthesis but are difficult to propagate long term. We examined cyclins A and B and p34cdc2 kinase, cell cycle regulatory proteins involved in the G2-M transition. Primary hepatocyte cultures were plated on laminin and stimulated with EGF to proliferate. Controls were unstimulated hepatocytes. In EGF treated cultures, the labeling index was >40% after 48 hours and >60% at 72 hours, but the mitotic index was <10%. Cyclins A and B and p34cdc2 kinase mRNA were three- to fourfold higher in EGF stimulated cultures than in controls. Immunoblotting also indicated the presence of cyclins A and B and p34cdc2 proteins, but the p34cdc2 kinase activity in EGF stimulated cultures was only twofold above controls after 48 hours and 72 hours in culture. Furthermore, immunohistochemical staining revealed nuclear localization of cyclins A and B at both 48 and 72 hours but p34cdc staining remained cytoplasmic. Hoechst staining of DNA revealed very few mitotic figures and several were abnormal (tripolar spindles). These results imply that hepatocytes are capable of undergoing DNA synthesis in vitro but are unable to complete the G2-M phase of the cell cycle.